ISBN 978-3-86727-
-Hordeum vulgare
L.) Grain
Y
ield Formation
Charles Mboya Matoka
Bacterial Community Responses to
Soil-injected Liquid Ammonium Nutrition
L.) Grain Yield Formation
Dokument1 15.01.2008 16:18 Seite 1
Cuvillier Verlag Göttingen
Cuvillier Verlag Göttingen
Cuvillier Verlag Göttingen
Cuvillier V
erlag Göttingen
and Effect of Temperature on Barley
(Hordeum vulgare
Matoka
C.
M. Bacterial Community Responses to Soil-injected Liquid
Ammonium Nutrition and Ef fect of Temperature on Barley ( 507 1
Institute of Plant Nutrition Justus-Liebig University Prof. Dr. Sven Schubert
BACTERIAL COMMUNITY RESPONSES TO SOIL-INJECTED LIQUID AMMONIUM NUTRITION AND EFFECT OF TEMPERATURE
ON BARLEY (Hordeum vulgare L.) GRAIN YIELD FORMATION
DISSERTATION
Submitted for the degree of Doctor of Agricultural Sciences (Dr. Agric. Sci.) to the Faculty of
Agricultural Sciences, Nutritional Sciences and Environmental Management
Justus-Liebig University Giessen, Germany.
By
Charles Mboya Matoka
from Rusinga Island, Nyanza, Kenya
Bibliografische Information er Deutschen ibliothek
Die Deutsche ibliothek verzeichnet diese Publikation in der Deutschen
Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar. Nonnenstieg 8, 37075 Göttingen Telefon: 0551-54724-0 Telefax: 0551-54724-21 www.cuvillier.de
Alle Rechte vorbehalten. Ohne ausdrückliche Genehmigung des Verlages ist es nicht gestattet, das Buch oder Teile
daraus auf fotomechanischem Weg (Fotokopie, Mikrokopie) zu vervielfältigen.
Gedruckt auf säurefreiem Papier 1. Auflage, 2008
CUVILLIER VERLAG, Göttingen 2008
1. Aufl. - Göttingen : Cuvillier, 2008
Zugl.: , Univ., Diss., 2007
978-3-86727-XXX-X
d Nationalb
Nationalb
Disputation was held on 21st December, 2007. Disputation commission members:-
Chairman: Prof. Dr. Steffen Hoy
Supervisors:
1. Prof. Dr. Sven Schubert
2. Prof. Dr. Sylvia Schnell
Examiners:
1. Prof. Dr. Joerg M. Greef
2. Dr. Yan Feng
Gießen
This project was conducted at the Institutes of Crop and Grassland Science and Agricultural Ecology, constituent Campuses of the Federal
Agricultural Research Centre (FAL), Braunschweig, Germany
In Collaboration with
TABLE OF CONTENTS
1.0 INTRODUCTION 1
1.1 Need for improved fertilization methods 1
1.2 Nitrogen forms taken up by crops 1
1.3 Limitations of nitrate based fertilizers 2 1.4 Agronomic requirements and economic importance of barley 2 1.5 Principle of CULTAN fertilization technique 3 1.6 Dilemma of inadequate and excess nitrogen nutrition 3 1.7 Potential of Nitrification inhibitor incorporation in CULTAN 4 1.8 Merits of CULTAN fertilization technique 4
1.9 Objectives of the study 5
2.0 MATERIALS AND METHODS 7
2.1 Determination of soil-injected liquid NH4+stability 7
2.1.1 Experimental site location and crop growth conditions 7
2.1.2 Experimental design 7
2.1.3 Nitrogen treatments and sampling intervals 8 2.1.4 Growth nutrient application and nitrification inhibitor incorporation 9 2.1.5 Crop sample categories at different intervals 11 2.1.6 Growth parameters, grain yield and yield components 11
2.1.7 Crop analyses 12
2.1.7.1 Chlorophyll analysis 12
2.1.7.2 Water-soluble carbohydrate (WSC) analysis 12 2.1.7.3 Total carbon and nitrogen concentrations 13
2.1.7.4 Nitrate analysis 13
2.1.7.5 Cation and anion analysis 13
2.1.7.6 Organic acid analysis 13
2.1.8 Soil analyses 14
2.1.8.1 Ammonium and nitrate determination 14 2.1.8.2 Potassium and phosphorus determination 14
2.1.8.4 Soil pH 15
2.1.9 Data analysis 15
2.2 Characterization of bacterial community responses to
CULTAN fertilization 16
2.2.1 Soil samples analyzed for bacterial occurrence 16
2.2.2 Bacterial DNA extraction from environmental soils 16 2.2.3 DNA amplification, purification and quantification 17 2.2.4 Single-strand conformation polymorphism (SSCP) technique 18 2.2.4.1 Reagents and equipments used in SSCP technique 18 2.2.4.2 Generation of the single-stranded DNA 18 2.2.4.3 SSCP gel silver staining and development 19 2.2.4.4 Band selection, excision and soaking 19 2.2.5 DNA cloning (ligation and transformation) 20
2.2.6 DNA Sequencing 21
2.2.6.1 Sequencing reagents and equipment 21
2.2.6.2 Sequencing procedure 21
2.2.7 Statistical Analyses 22
2.2.7.1 Digital image analysis 22
2.2.7.2 Sequence analyses 22
2.3 Evaluation of the biodiversity of ammonia oxidizing bacteria 24
2.3.1 Detection of ammonia oxidizing bacteria (AOB) 24
2.3.2 Selection and optimization of amoA primers 24
2.3.3 Selection of AOB for use as positive control 24
2.3.4 PCR amplification AOB with amoA primer 24
2.3.5 Development of amoA SSCP gels 25
2.3.6 Band selection, ligation, transformation and sequencing 25
2.3.7 Statistical analysis 25
2.4 Ammonia oxidizing bacteria population abundance in
CULTAN-fertilized soils 26
2.4.1 Quantification of N. multiformis gene copies 26
2.4.2 Real-Time PCR optimization 26
2.4.3 Generation of amoA standard curves 27
2.4.4 Threshold cycle determination 27
2.4.5 Melting point curves of amoA genes 27
2.4.5 Statistical analysis 27
2.5 Effect of temperature on CULTAN-fertilized barley 28
2.5.1 Experimental site and crop growth conditions 28
2.5.2 Experimental design 28
2.5.3 Temperature and nitrogen treatments 28
2.5.4 Soil sampling zones 29
2.5.5 Carbon Exchange Rates (CERs) and SPAD measurements 31
2.5.6 Shoot and root biomass estimates 31
2.5.7 Crop analyses 32
2.5.8 Soil analyses 32
2.5.9 Data analysis 33
3.0 RESULTS 34
3.1 Stability of soil-injected liquid NH4+ 34 3.1.1 Soil physical and chemical characteristics 34 3.1.2 Concentration of soil-injected NH4+ 36 3.1.3 Concentration of NO3-in the soil 37 3.1.4 Potential of nitrogen loss in CULTAN-fertilized soils 38
3.1.6 Phosphorus and potassium availability under CULTAN 42
3.1.7 Crop growth and yield responses to CULTAN-fertilization 44
3.1.7.1 Biomass accumulation and relative growth rates (RGRs) 46
3.1.7.2 Barley grain yield and yield-forming factors 48
3.1.8 Chemical composition of crops fertilized with different N forms 50
3.1.8.1 Crop nitrogen uptake and partitioning 50
3.1.8.2 Shoot mineral concentrations 52
3.1.8.3 Leaf chlorophyll and carotenoid concentrations 55
3.1.8.4 Sugar concentrations 57
3.1.8.5 Organic acid concentrations 58
3.2 Bacterial community diversity responses to CULTAN fertilization 60
3.2.1 Bacterial community detection 60
3.2.2 Spatial dynamics of detected bacterial communities 60 3.2.3 Temporal dynamics of detected bacterial communities 64 3.2.4 Characterization of CULTAN-associated bacterial communities 66
3.2.5 Composition of CULTAN-associated bacterial communities 66
3.2.6 Phylogenetic relationships of characterized CULTAN bacterial groups 67
3.3 Biodiversity of AOB associated with CULTAN-fertilized soils 69
3.3.1 Spatial changes of ammonia oxidizing bacteria under
CULTAN fertilization 69
3.3.2 Temporal dynamics of ammonia oxidizing bacteria under
CULTAN fertilization 71
3.4 Estimate of AOB population abundance in
CULTAN-fertilized soils 75
3.5 Growth temperature effect on grain yield of
CULTAN-fertilized barley 79
3.5.1 Barley growth duration and shoot height 79 3.5.2 Biomass accumulation and partitioning 80
3.5.3 Grain yield and yield forming-factors 81
3.5.4 Response of yield-forming factors to temperature
and CULTAN-fertilization 83
3.5.5 Carbon exchange rates (CERs) and SPAD 83
3.5.6 Soil and crop nutrient concentration 85
4 DISCUSION 89
4.1 Stability of soil injected liquid NH4+ 89
4.1.1 Establishment of NH4+sorption-complex zones 89 4.1.2 Nitrogen forms available in CULTAN-fertilized soils 92
4.1.3 Barley root growth responses to CULTAN fertilization 94 4.1.4 Barley aerial growth responses to CULTAN fertilization 96 4.1.5 Possible mechanisms involved in mixed N nutrition under CULTAN 99
4.2 Bacterial community responses to CULTAN-fertilization 102
4.2.1 Occurrence of bacterial communities within CULTAN-fertilized soils 102 4.2.2 Functional responses of AOB in CULTAN-fertilized soils 103 4.2.3 Effect of bacterial communities on N form in CULTAN fertilized soils 104 4.2.4 Bacterial community shift occurrence in CULTAN fertilized soils 105 4.2.5 Possible influences of soil pH on microbial community 107
4.2.6 Bacterial community structure restoration from CULTAN-effects 108
4.2.7 Potential use of nitrification inhibitors to suppress AOB activity in
CULTAN fertilization 109
4.3 Ammonia oxidizing bacteria population abundance in
CULTAN-fertilized soils 111
4.3.1 Relative AOB population abundance in CULTAN fertilized soils 111
4.3.2 Potential of ammonia oxidation by non-Proteobacteria 112
4.4 Effect of growth temperatures on CULTAN-fertilized
Barley crop grain yield formation 114
4.4.1 Effects of temperature on CULTAN-fertilized barley growth responses 114
4.4.2 Temperature effects on grain yield of CULTAN-fertilized barley 114 4.4.3 Fate of CULTAN-fertilizer upon injection into soil 115 4.4.4 Potential of CULTAN-fertilization for crop production 116 4.4.5 Possible effects of temperature on CULTAN-fertilization 117
5. SUMMARY 119 6. ZUSAMMENFASSUNG 121 7. REFERENCES 124 8. ACKNOWLEDGEMENT 143 9. DEDICATION 145 10. CURRICULUM VITAE 146
1.0 INTRODUCTION
1.1 Need for improved fertilization methods
Nitrogen (N) is one of the nutrients required for plant growth since it forms an integral part of various vital compounds. Upon uptake, it is may be assimilated into plant tissues and organs. Normally, nitrogen accounts for about 2-5% of the total plant dry matter. For this reason, it is usually required in appreciably larger amounts than the other nutrients (Marschner, 1995). The use of commercial fertilizers, especially nitrogen aims at crop growth and yield improvement. However, losses associated with the currently employed conventional fertilization methods calls for the adoption of alternative methods or improvement of the existing ones. The application of liquid ammonium fertilizer is emerging as a noble fertilization technique because it mitigates nitrate associated losses.
1.2 Nitrogen forms taken up by crops
In soil, nitrogen occurs both as organic and inorganic compounds, with 95% or more being organic (Miller and Cramer, 2004). The apparent inadequacy of nitrogen availability for uptake by crops necessitates the application of commercial fertilizers and has made it a common agricultural practice. Generally, nitrate and ammonium are the
main inorganic nitrogen sources taken up by roots of higher plants. Upon uptake, NO3
-can be reduced either within the root or shoot and excess amounts -can be stored in different plant tissues like vacuoles (Marschner, 1995). Since NH4+ is usually toxic to
plants when taken up in large amounts, it is thus a prerequisite that crops to which it is exposed must be well adapted to either prefer or tolerate its effects (Gerendas et al.,
1997). Crops fertilized by NH4+ should be capable of regulating both internal and
external acidic pH conditions to avoid ‘ammonia syndrome’ resulting from ionic
imbalances (Mehrer and Mohr, 1998). Because of its toxicity effects on crops, NH4+ is
normally assimilated within the roots (Britto and Kronzucker, 2002). The application of
urea based fertilizer which upon hydrolysis confers NH4+ related characteristics has
recently been gaining popularity (Miller and Cramer, 2004). However, mixed N nutrition occurring in the presence of both NH4+ and NO3- is capable of supporting better crop
1.3 Limitations of nitrate based fertilizers
Nitrate availability in soil is greatly constrained by its high mobility rate which facilitates its rapid loss. Its loss mainly occurs through leaching and denitrification which reduces its availability within the rhizosphere. In nature, nitrification process whose end
product is NO3- thus leads to appreciable N losses from agricultural land. Besides
reducing nutrient availability for uptake, NO3- leaching potentially pollutes underground
water bodies and also cause eutrophication of above ground water bodies through run-off. It is for these reasons that the current farming practices emphasize improved crop yields in a benign environment through a sustainable production system. The main purpose of improved farming method is to enhance food security to feed the ever increasing human po- pulation (Mangelsdorf, 1966; asil, 1998). This undertaking has opened up a window of research opportunity for the re-evaluation of previous agricultural practices. This is crucial since cereal production which provides the bulk of edible fibre consumes more than 60% of the total N fertilizers worldwide annually (Rao and Popham, 1999).
1.4 Agronomic requirements and economic importance of barley
Barley is a member of the grass family, Poaceae. It is a grain crop that is currently grown in more than 100 countries worldwide and is ranked fourth both in terms of quantity and area of production covered by cereals. It grows best on light soils rich in minerals with moderate water requirements. It is tolerant to cold stress and resistant to soil salinity than wheat. It exists either as winter or spring crop and its cultivars largely occur as six or two row grained ears. It serves as human food such as bread and other cereal products besides its usage as livestock fodder while green or hay when dry and silage in the conserved form. Dry straw can be used as animal bedding while grains can also be processed into animal feed concentrates. Several industrial brews such as beer, whiskey and malt syrup are among important alcoholic drinks manufactured from barley which are consumed by a large human population. The crop generates a lot of income to economies of countries such as USA, China, Canada, United Kingdom, Germany and Netherlands among others that produce and process its products (Foster, 1981).
1.5 Principle of CULTAN fertilization technique
The new fertilization method is based upon the injection of concentrated liquid NH4+ into soil and its subsequent adsorption onto clay particles and soil organic matter
which enhance its long-term availability for crop uptake (Sommer, 1995). The fertilization method is developed within the tenets of emerging crop production paradigm, which emphasize improved crop yields in a sustainable manner. The adoption of such agricultural practice does not compromise ecological and environmental quality and it plausibly sustains and improves crop yields. When adsorbed onto clay soil matrix,
NH4+ forms a sorption-complex which serves to regulate its uptake by inhibiting root
growth and penetration into the concentrated zone (Zhang and Rengel, 2000). In Germany, the fertilization technique is very popular and is referred to as CULTAN. The acronym ‘CULTAN’ is an abbreviation of Controlled Uptake Long Term Ammonium Nutrition (Sommer, 1993; 2000; 2003). Similar methods are being practised elsewhere, though they are known by different names. For example, it is referred to as point injection in Canada and America (Janzen et al., 1991) while in Asia, more specifically Japan, sulphur-coated nitrogen are applied as slow release fertilizers and are commonly referred to as Controlled Nitrogen Release Fertilizers (CNRF) (Wakimoto, 2004). Adoption of the
CULTAN fertilization method promotes localized NH4+deposits within the rooting zone
whose toxicity causes root inhibition into the injection-point whereas the less toxic peripheral zones promote intensive root growth network (Sommer, 2000).
1.6 Dilemma of inadequate and excess nitrogen nutrition
In most tropical soils, N is insufficient for satisfactory crop yield outputs. On one hand, the use of fertilizer inputs in developing countries, especially in sub-Saharan Africa, is quite low with essential nutrients often inadequately supplied due to high input costs, unavailability and poor marketing infrastructure (Dakora and Keya, 1997). On the other hand, developed countries are faced with environmental pollution resulting from excessive amounts of NO3- fertilizer applied in an effort to improve crop yields (Jeuffroy
et al., 2002). Whereas developing countries are mainly faced with fertilizer inadequacy,
developed countries are experiencing excessive fertilizer application problems with the potential of causing environmental pollution. A middle ground for both the developed and developing countries may be struck through improved fertilization techniques such as
CULTAN technique which has the ability of improving N availability for crop uptake during the cropping period. When fine-tuned, the fertilization technique may alleviate constraints associated with fertilizer input inadequacies of developing and excesses experienced in the developed worlds.
1.7 Potential of nitrification inhibitor incorporation into CULTAN
A potential N loss mitigation method that would be compatible with CULTAN fertilization technique is the incorporation of nitrification inhibitors into soil injected ammonium. Nitrification inhibitor (NI) incorporation into ammonium fertilizer can
temporarily improve nitrogen retained as NH4+ through the suppression of the
nitrification process (Crawford and Chalk, 1993). Application of NI influences not only fertilizer efficiency by reducing leaching or denitrification losses, but also the ratio of available inorganic N forms (Vanneli and Hooper, 1992). Occasionally, immobilization of N increases in response to NI-incorporation into soil thereby reducing N available for crop uptake (Vanneli and Hooper, 1993). However, the potential benefits accruing from NI-incorporation into NH4+ fertilizer through the suppression of nitrification supersedes
the corollary effect of N losses when no nitrification inhibitor is incorporated. One of the commercially available nitrification inhibitors reported to successfully reduce nitrogen losses through less gas emissions is Nitrapyrin® (McCarty, 1999).
1.8 Merits of CULTAN fertilization technique
The popularity of CULTAN fertilization technique is due to the merits associated with its application. The method bases upon NH4+ as the dominant N form, which inhibits
root penetration through the injection depot. This occurs because of its toxicity to roots. Proliferation of intensive root network around the injection zone is thought to trap diffusing ions, while the root growth inhibition by free ions within the injection-point offers a self-regulatory mechanism for N uptake as opposed to conventional nitrate fertilizer application that is made in splits to coincide with crop growth stages deemed by the farmer to require increased nutrient supply, which may, however, not be the case based upon the
internal crop nutrient status (Sommer, 2000). Since NH4+ assimilation within the crop
regulatory measure because the uptake has to be determined by the proportion of C-skeleton translocation to the roots (Cramer and Lewis, 1993). The resulting organic nitrogenous compounds are thus incorporated into root system or channelled to other parts including the shoot. The supply of C-skeletons through photosynthesis and translocation of photosynthate to the root in conjunction with the redistribution of root assimilated nitrogenous compounds creates a counter-current trafficking of the growth essentials in opposite directions which result in a source-sink relationship for different products that are highly dependent upon each other. Along side these physiological merits, the advantages attained through the use of mechanized farm equipments which facilitate the wide adoption of CULTAN fertilization technique in large scale and in different crops are some of the factors that have contributed to its popularity.
1.9 Objectives of the study
The study focused on generating information to bridge the knowledge gap between crop and microbial responses in CULTAN-fertilized soils. In addition, crop growth and yield responses resulting from the interaction effects of CULTAN fertilization and different growth temperatures have not been evaluated to date. To be able to exploit the full potential of CULTAN-fertilization method, its interaction with abiotic and biotic factors need to be addressed. Currently, various mechanisms and processes relating to CULTAN-fertilization are not well understood and some are at best only assumed. Though it is known that NH4+adsorbs onto clay particles and soil organic
matter to form sorption-complex within the injection-point (Sommer, 2000), it is however, not known how stable the injected NH4+is within the soil. In case the injected
NH4+is stable to any extent, it is unknown for how long it would support crop growth.
Such information on NH4+stability can help in decision making whether a single injection
at a certain concentration would be sufficient to support crop growth throughout the entire season or whether there would be need for multiple injections to be performed at intervals coinciding with particular crop growth stages. Therefore, the first objective of this study was to evaluate the stability of soil-injected liquid ammonium and crop growth responses.
No data is available to support the view that highly concentrated NH4+is toxic to
soil microbes, especially bacteria. This presumption seems to stem from previous reports highlighting root growth inhibition by concentrated NH4+ fertilization (Sommer, 2000).
Chemolithoautotrophic bacteria are known to use ammonia as an energy source during oxidation when they transform ammonia to nitrate via nitrite (Purkhold et al., 2000). There is however, no experimental data to illustrate bacterial community responses to soil-injected liquid NH4+. In light of this information gap, the second objective of the
study focused on the assessment of the occurrence and response of bacterial communities associated with soil-injected liquid NH4+analyzed by targeting 16S rRNA gene. Attempts
to detect occurrence and abundance of ammonia oxidizing bacteria (AOB) while targeting ammonia monooxygenase subunit A (amoA), functional gene, were also made.
The direct influence of temperature on plant growth caused by effects on root mineral nutrient and water uptake as well as translocation is well known (Macduff and Jackson, 1991). In addition, the effect of temperature on microbial activity, particularly ammonia oxidation rates has been reported (Avrahami et al., 2003). However, no study has been conducted to elucidate the interaction of the two factors under a cropping
system fertilized through the CULTAN method, which is predominated by NH4+ over
NO3-. There is a high possibility of growth temperatures and N form available for
absorption by crops interacting with each other, especially in the presence of mixed N nutrition resulting from the nitrification of soil-injected NH4+ to cause a suite of growth
responses with strong implications on grain yield and yield forming factors. In this regard, the last objective of this study was to evaluate the impact of liquid NH4+ on barley
yield and yield forming factors under different growth temperatures. In summary, the following specific objectives were addressed by the
study:-i) To evaluate stability of CULTAN-injected NH4+and its effect on barley crop growth.
ii) To assess occurrence and characterize bacterial community responses to CULTAN fertilized soils.
iii) To investigate occurrence and abundance of ammonia oxidizing bacteria (AOB) in CULTAN-fertilized soils.
iv) To elucidate the interaction effects of soil-injected NH4+and growth temperatures on
2 MATERIALS AND METHODS
2.1 Determination of soil-injected liquid NH4+stability
2.1.1 Experimental site location and crop growth conditions
The experiment was performed at Federal Agricultural Research Centre (FAL), Braunschweig, Germany. This was a pot experiment performed in three solar-irradiated growth chambers supplemented with an artificial lighting system. Each chamber measured 10 m long, 8 m wide and 10 m high with a glass roof slanting at 45o. Illumination was mainly solar, but artificial light from lamps provided irradiation at 400 μmol m-2s-1 after the first month of sowing. Spring barley (Hordeum vulgare L.) cv. Maresi procured from Lochow-Petkus GmbH was chosen as a model cereal crop for the study. The containers were black polyvinyl with a capacity of 90 L and filled with 80 L soil. Each container had a diameter of 54 cm and was subdivided into four equal segments (quadrants) with a sowing area of 0.0572 m2 to support 22 seedlings sown at a depth of 2.5 cm within an inter- and intra-row spacing of 5 cm. The containers were placed in wooden troughs covered with black plastic sheet holding irrigation water (Fig. 9a).
The containers had five 1 cm diameter irrigation holes on the lower side of each quadrant for irrigation water percolation into the soil. The base of each quadrant was filled with 2 cm layer of pebbles for easy water entry. Crops were irrigated from the bottom once weekly with 5 L for 2 h in the first month and 10 L were administered for 2 h twice a week during the rest of the growth period. Excess irrigation water was drained by hand-held pump and reutilized in subsequent irrigations. Fortnightly, 1 L of the drained water was sprinkled onto the soil surface to improve the moisture content. Irrigation water was contained in wooden troughs measuring 1.2 m long, 1.2 m wide and 0.08 m high and placed on mobile trolleys adjusted to 0.8 m high. Photoperiod was 14/10 h, day/night, respectively. Irradiation lamps were switched off 1 month after sowing. Relative humidity ranged between 55 – 75% while growth temperatures were 20/15 oC, day/night, respectively, throughout the season.
2.1.2 Experimental design
The experimental arrangement was a Randomized Complete Block Design (RCBD), comprising six nitrogen treatments. Each container represented a treatment while each
quadrant was a replication. Each treatment had four replications (quadrants) (Fig. 1). The six nitrogen treatments comprised four NH4+-based treatments while the other two were comparison checks fertilized with nitrate or non-fertilized control. Crops were sampled destructively at eight different growth stages as described in the next section.
2.1.3 Nitrogen treatments and sampling intervals
Crop and soil samples from each treatment were collected at eight different intervals. Out of the six nitrogen treatments included, four were NH4+-based in addition to nitrate and non-fertilized control. Two of the four NH4+ treatments were incorporated with nitrification inhibitors {NI}, (Nitrapyrin®) at a rate of 5 and 20%, designated as (NH4+ + 5%NI + Crop) and (NH4+ + 20%NI + Crop), respectively. The other two NH4+ treatments without NI incorporation comprised cropped, (NH4+ - NI + Crop) and uncropped treatment (NH4+ - NI - Crop). Each of the six treatments was sampled at eight different intervals with each coinciding with a specific crop growth phase. The first was at seedling growth stage (ZS 15) coinciding with 11 days after sowing (DAS), while the next three stages occurred at tillering stage, which was sub-divided into three distinct stages (ZS 21, 25 and 29) which coincided with 25, 30 and 39 DAS. The fifth sampling was carried out at stem elongation (ZS 36), which coincided with 60 DAS, whereas the sixth sampling stage performed at booting stage (ZS 45) coincided with 66 DAS. Seventh sampling was done at medium milk kernel stage (ZS 75) coinciding with 80 DAS and eighth sampling interval was performed at crop maturity (ZS 99), which coincided with 109 DAS. Developmental stages were based on Zadoks’ growth scale (ZS) (Zadoks et al., 1974). After the sixth sampling, treatments awaiting the next two sampling schedules were rearranged into a single growth chamber. Season two sampling was performed similarly to those at season one. A summarized list of the six treatments is hereunder:-
(i) NO3- =N-form; Ca(NO3-)2 applied at 4g N container as a single application [T1]
(ii) NH4+ = NH4+ without Nitrapyrin®, but cropped, (NH4+ - NI + Crop); [T2]
(iii) NH4+= NH4+ with 5% Nitrapyrin® and cropped, (NH4+ +5% NI + Crop); [T3]
(iv) NH4+ = NH4+ with 20% Nitrapyrin® and cropped, (NH4+ +20% NI + Crop); [T4]
(v) NH4+ = NH4+ without Nitrapyrin® and uncropped, (NH4+ - NI - Crop); [T5]
2.1.4 Growth nutrient application and nitrification inhibitor incorporation
Soil used in the experimentation was from three portions constituted by sub- and two separate top- soils mixed in ratios of 1:1:1, respectively. Nitrate was provided as Ca(NO3-)2 while NH4+ was applied as diammonium phosphate (DAP), (NH4+)2HPO4. Each treatment received 4 g N of either NO3- or NH4+. Nitrate was sprayed whereas NH4+ was injected at the centre of each quadrant. Injection was performed at a depth of 7 cm using 1 cm diameter aluminium rod. The holes were fitted with 20 ml Eppendorf tubes to simulate spoke wheel injectors used for CULTAN injection under field conditions (Fig. 2a and b). After injection, the holes were refilled with soil and marked with thin wooden pegs. Additional 18 g P in the form of Ca(H2PO4)2.H2O was applied in nitrate and non-fertilized treatments to balance out the P contributed by DAP fertilizer. Essential nutrients whose effects were not being evaluated were adequately supplied by mixing into soil so as to ensure availability of sufficient mineral nutrition. Macro nutrients were provided as P = 5 g, K = 13 g, Mg = 27 g, Ca = 6 g per treatment, whereas micro-nutrients too were applied as Fe = 25 mg, Mn = 10 mg, Cu = 5 mg, B = 3 mg, Zn = 5 mg and Mo = 0.5 mg per treatment. Incorporation of the two Nitrapyrin levels, 5 and 20% into two of the ammonium treatments was applied after its dissolution into toluene and acetone in seasons one and two, respectively.
Fig. 1: A diagram showing both vertical and lateral soil sampling zones in two of the quadrants. Zone 1 is situated 2 cm below the surface while 2a was at 7 cm depth and was coplanar with 2b and 2c though at different circumferences. Zone 3 was 15 cm below the surface or 8 cm below the injection point and finally zone 4 was at a depth of 27 cm.
Fig. 2a and b: A diagram showing spoke wheeled liquid NH4+ injection equipment (a) and ammonium injection shares (b) with pressurized valves to release the liquid fertilizer during field operation
7-(4) 7-(2c) 2b 2c 2b 2c 2b 2c 2c 2c 2c (2b) (2a) (1) (3) 7-(4) 7-(2c) 2b 2c 2b 2c 2b 2c 2c 2c 2c (2b) (2a) (1) (3) 7-(4) 7-(2c) 2b 2c 2b 2c 2b 2c 2c 2c 2c (2b) (2a) (1) (3)
2.1.5 Crop sample categories at different intervals
Crop stand of 22 was sustained in each quadrant, out of which seven were edge plants and the remaining 15 were used for growth measurements and analyses. The first harvest performed at 11 DAS comprised 15 seedling shoots used for biomass and ion determination. Second sampling performed at 25 DAS was sub-divided into two, with the first category comprising 10 plants processed for biomass, carbohydrate, total nitrogen and nitrate concentration analyses, whereas the second category of 5 plants, which were fractionated into main and minor tillers had each of the five main tillers further fractionated into stem and different leaves. First fully open leaf of the main tiller was harvested, but not used for any analysis. The two successive leaves below uppermost open leaf were pooled together and used to determine the concentration of organic acids. Fourth leaf from the five main stems were pooled and used for chlorophyll concentration determination. Leaf samples were packed into aluminium containers filled with liquid nitrogen and transferred into -80 oC freezers. Frozen leaf samples were ground in cooled pestle and mortar filled with liquid nitrogen. Samples from 25, 30, 39 and 60 DAS were used for chlorophyll measurements in season one, whereas season two had an extra interval sampled at 66 DAS. The organic acid concentrations were determined from leaf samples harvested at 30, 39 and 60 DAS. Whole plants were used to determine biomass and relative growth rates of samples dried in the oven at 105 oC for 48 h. Water soluble carbohydrates were analyzed from 30, 39, 60 and 66 DAS in both seasons. Sub samples were used to determine ion concentration and total ash. The crop samples were ground using Brabender laboratory mill with 1 mm grid.
2.1.6 Growth parameters, grain yield and yield components
Barley crop growth and yield responses to different N-forms were compared. The samples harvested at the eighth stage (109 DAS) were used for grain yield estimates. Out of the possible 22 barley plants per quadrant, seven were edge plants and the remaining 15 were considered for yield estimates. Crop growth duration until maturity was recorded. Shoot heights were estimated while tiller numbers were counted also. Surviving plant population at harvest was counted. Total number of tillers was counted and the average number of tillers calculated. Fertile ear numbers were counted and the number of grains per ear averaged. Ear and shoot samples were oven dried at 60 oC for 48 h and ground for chemical analysis. The
dried grains were threshed, winnowed, weighed and counted. Thousand grain weights were determined by multiplying average weights of three replicates of 250 grain weights by four.
2.1.7 Crop analyses
Crop samples were analyzed for a number of parameters as described below. Biomass was determined from sub-samples of five non-fractionated whole plants that were oven dried at 105 oC for 48 h. Total ash was determined by combusting the crop samples at two phases, initially at 185 oC for 90 min and then secondly at 320 oC for a similar period.
2.1.7.1 Chlorophyll analysis
Chlorophyll concentration was determined from the fourth leaf harvested from the main tiller. The leaf samples were preserved in liquid nitrogen immediately after harvest then transferred to -80 oC freezer. They were later powdered in frozen pestle and mortar filled with liquid nitrogen during grinding. The pigments were extracted in 80% acetone as detailed in (Schittenhelm and Menge, 2006) after which chlorophyll a and b absorbance were spectrophotometrically determined at 647 nm and 663 nm, respectively, while that of carotenoids were determined at 470 nm. Pigment concentrations were calculated using the formula developed by Lichtenhaler (1987).
2.1.7.2 Water-soluble carbohydrate (WSC) analysis
Water soluble carbohydrates (WSC) were determined using high performance liquid chromatography (HPLC) as described by Schittenhelm (1999). In brief, about 0.02 g of dry plant material was mixed into 1 mL distilled water vortexed and incubated for 7.5 min in a shaker set at 80 oC warm water bath. After the initial incubation, the tubes were removed, mixed by inverting and once again reincubated for 7.5 min in 80 o C water bath. The samples were cooled at room temperature, centrifuged and then pipetted into 2 mL tube. A repeat extraction performed and extracts were pooled, filled into 1.2 mL HPLC glasses and compared with standards using a computer integrated with a differential refractometer.
2.1.7.3 Total carbon and nitrogen concentrations
Total nitrogen and carbon were analyzed through the DUMAS method from 0.2 g using an automated FP-2000 LECO analyser (LECO Corporation, St. Joseph, MI, USA). The equipment comprises an automatic sampler, combustion oven (1049 oC) and analyzer. Nitrogen was measured by the warm conductivity detector (WCD) whereas carbon was
2.1.7.4 Nitrate analysis
Crop sample nitrate concentration was analyzed using colorimetric SKALAR method (Skalar-Analytik GmbH, Erkelenz, Germany). In summary, about 0.5 g of dried crop samples or 0.2 g of frozen material was extracted in warm water and vortexed. The supernatant was used for nitrate and ammonium analysis using Skalar analyzer.
2.1.7.5 Cation and anion analysis
About 3 g of ground plant material was extracted for 90 min in 150 ml calcium-lactate solution, filtered and used for potassium, calcium, magnesium and sulphur using atom absorption spectrophotometer (AASP) set on omission mode. Phosphorus was analyzed from the same extract using spectrophotometer, Zeiss-Braun-Analysenstrasse model, Germany at an extinction of 720 nm. Chloride was analyzed using ion chromatography (761 compact IC – Metrohm Ionanalytik, AG CH-9101 Herisaue, Sweden), following the method described by Small et al. (1975).
2.1.7.6 Organic acid analysis
Frozen leaf samples (0.2 g) were extracted twice for 30 min in 1 m boiling distilled water, then centrifuged for 10 min at 20,000 x g. The supernatant was pooled and analyzed for organic acids using high performance liquid chromatography (HPLC) by adopting a Rezex ROH organic acid H+ column run on 0.005 M sulphuric acid in 30 oC at a flow rate of 0.5 ml min-1. The detection of the organic acid was achieved using a Shodex RI-TI refractive index detector (Showna Denko, K. K. Tokyo, Japan).
L measured by an infrared detector (IRD).
2.1.8 Soil analyses
At each sampling schedule, soil samples were collected from six different zones consisting of four vertical zones 1, 2a, 3 and 4 and two lateral zones comprising 2b and 2c per replication in each NH4+ treatment. Only the four vertical zones were considered in nitrate and non-fertilized treatments (Fig. 1). Zone 1 was 2 cm below the soil surface while zone 2a with a radius of 2.5 cm was located at a depth of 7 cm. Zones 3 and 4 were at depths of 15 and 27 cm, respectively, below the soil surface along the vertical column. Zone 2b formed a ring around zone 2a with a breadth of 2.5 cm while the outermost ring surrounding 2b was 2c and was 2.5 cm wide (Fig. 1). Each zone was sampled up to a depth of 3 cm. Six of the eight soil sampling intervals were analyzed. They included 11, 25, 30, 39, 60 and 109 days after fertilization (DAF) in the first season. In the second season, only five of the six sampling intervals were analyzed. Interval 39 DAF was omitted. Root development around the injection-points were scored on a scale of 0 to 3, depicting; 0 = none depot existence, 1 = slight depot formation, 2 = moderate and 3 = well formed depots.
2.1.8.1 Ammonium and nitrate determination
Soil sub samples were homogenized by sieving through a 5 mm mesh from which 25 g was weighed into a 250 mL conical flask and mixed with 100 mL of 0.0125 M CaCl2. The soil samples were then placed on rotary shaker to extract for 1 h then paper-filtered. The filtrate was used for the determination of NO3- and NH4+
colorimetric SKALAR method (Skalar Analytik GmbH Erkelenz, Germany).
2.1.8.2 Potassium and phosphorus determination
Extractable P was analyzed using spectrophotometer at an extinction of 720 nm, whereas K was determined through emission mode of atomic absorption spectrophotometer (AASP). The procedure was similar to that described for crop samples in section 2.1.7.5.
2.1.8.3 Cation exchange capacity
Cation exchange capacity (CEC), soil structure and texture were determined at the Agricultural and Environmental Research Centre (LUFA), Hannover, Germany. The cations were analyzed using spectrophotometer while sand, clay and humus were also determined.
2.1.8.4 Soil pH
Soil pH was determined through the adoption of VDLUFA (1991) method where 10 g of air dried soil was sieved through a 2 mm mesh and weighed into 100 ml conical flask to which 50 mL 0.01 M CaCl2 solution was added, stirred to mix using a rotary glass rod then left to extract for 1 h. The extract was used to measure soil pH using a pH meter.
2.1.9 Data analysis
Statistical analysis was performed using statistical analysis system (SAS) for windows program version 9.1 based upon the general linear model (GLM). Multivariate analysis of variance (ANOVA) was performed to determine and compare treatment differences between the two seasons and among the treatments. The data presented in this section are treatment averages for each separate season. Where applicable, zonal differences were analyzed and differences between sampling intervals were compared among soil samples of the six treatments. The uncropped NH4+ treatment had no crop information since it was not sown. Nitrate and non-fertilized treatments had only soil samples along the vertical section since zones 2b and 2c were not considered. Similarly, crop biomass estimates at different growth stages, chlorophyll, total nitrogen, NH4+ and NO3 -concentrations as well as the water soluble carbohydrate -concentrations were subjected to multivariate analysis to compare the differences among subsequent harvest intervals in each season and the overall of the two seasons. One way ANOVA was also performed to compare the mean yield differences among the nitrogen treatments. The means were considered significant if P 0.05 and post-ANOVA was performed using Tukey test to determine the level of statistical significance. Where appropriate, correlation analyses were performed. The inclusion of non-fertilized control enabled the assessment of N fertilization effect, while the effect of NH4+-based fertilizers on crops was assessed by comparing cropped and uncropped treatments. Additionally, nitrate treatment provided a basis for the comparison of the two nitrogen forms whereas the influence of nitrification inhibitor (NI) incorporation of two different concentrations were also included for comparison against treatments without NI incorporation and incorporation of low and high NI concentrations.
2.2 Characterization of bacterial community responses to CULTAN fertilization
Soil samples from which bacterial DNA was extracted were five of the six treatments described under ammonium persistence ev aluation. Ammonium treatment incorporated with 20% NI was omitted. Details of the treatments are described in section 2.1.3.
2.2.1 Soil samples analyzed for bacterial occurrence
Soil samples harvested in the first season were used for analysis. The soil samples used for molecular microbiological investigations were sampled at 30, 60 and 109 DAF. Briefly, three of the treatments were NH4+ -based out of which, two were not incorporated with NI. The treatments were cropped NH4+ without NI presented as (NH4+ - NI + Crop), uncropped designated as (NH4+ - NI -Crop) and cropped NH4+ treatment incorporated with 5% NI was designated as (NH4+ + 5%NI + Crop). Nitrate and non-fertilized control treatments were included as comparison checks. To assess ammonium nutrition spatial diffusion effects on bacterial communities, both lateral and vertical zones were considered. Only two vertical zones were chosen in nitrate and non-fertilized control treatments and no lateral zones were considered. Zones along the vertical section included 2a and 3 located at depths 7 and 15 cm below soil surface, respectively (Fig. 1). Zone 2c also located at a depth of 7 cm was coplanar to zone 2a across the lateral section though separated from it by 2.5 cm radius distance. The breadth of each zone was 2.5 cm and sampling depth was 3 cm. Three replications were considered for each sample collected at 30, 60 and 109 DAF for each treatment and zone (Fig. 1).
2.2.2 Bacterial DNA extraction from environmental soils
Genomic bacterial DNA was extracted from soil samples described above using Bio101 systems, FastDNA® spin kit for soil (QBiogene, USA). In summary, 0.5 g soil was weighed into screw-cap tubes with ceramic and silica-beads (Roth Karlsruhe, Germany). Buffer (122 MT) was added to the soil to facilitate cell disruption with the help of FastPrep® instrument (model FP120, QBiogene, USA) operated at a speed of 5.5 for 30 s after securing the screw cap tubes during processing. The tubes were centrifuged at 14,000 x g for 5 min after which the extract was transferred into 1.5 ml tube. The resulting pellet was resuspended in 950 μL sodium phosphate buffer to which 250 protein precipitating solution (PPS) was added and mixed by hand shaking then centrifuged at 14,000 x g for 5 min. About 1 mL
binding matrix suspension solution was added and the tubes were inverted by hand for two minutes to enhance binding of DNA to matrix and placed on a rack to settle for 3 min. About 700 μL of the upper supernatant portion was carefully discarded and the remaining 600 μL pippeted into a SPIN Filter and centrifuged at 14,000 x g for 1 min. The catch tube was emptied and the remaining supernatant was refilled and the process repeated. The DNA was washed with 500 μL of salt-ethanol-wash-solution (SEWS-M) and centrifuged at 14,000 x g for 1 min. The flow-through was discarded and filter replaced in the same catch tube and centrifuged once again at 14,000 x g for 2 min. The washing step was repeated to remove co-extracted contaminants. Finally, the filters were removed and replaced in fresh catch tubes, air dried for 5 min at room temperature and eluted with 60 μL of (TE) Tris-EDTA-buffer. About 40 μL DNA aliquots were stored at -20 oC while 20 μL was used for immediate analysis. The purity of extracted DNA was analyzed by electrophoresis on 1.0% agarose gel and photographed using BioDocAnalyze (Biometra Goettingen, Germany). DNA quantification was achieved using a fluorescent dye, PicoGreen (MoBiTec., Goettingen, Germany) and the measurements were performed on Labsystems Fluoroskan II (GMI, Albertsville, Minnesota, USA) at a wavelength of 485 nm emitted at 530 nm.
2.2.3 DNA amplification, purification and quantification
Amplification of genomic bacterial DNA was achieved through adoption of polymerase chain reaction (PCR) procedure, an in vitro process that employs a heat-stable polymerase enzyme. Forward and reverse universal bacterial primers targeting 16S rRNA genes were used. Forward primer (Com1), (5'-CAGCAGCCGCGGTAATAC-3') targeted positions 519 to 536 while reverse primer (Com2+Ph), (5'-CCGTCAATTCCTTTGAGTTT-3') targeted positions between 907 and 926. Cycling was performed on a thermocycler (model Primus 96plus, MWG Biotech, Ebersberg, Germany) in 50 μL micro-tubes (Flat Cap Micro Tubes). The reaction mixture comprised 1× PCR buffer containing 1.5 mM MgCl2, deoxynucleoside triphosphate solution (dNTPs) of 0.2 mM per tube. Forward and reverse primers were added at a rate of 0.5 μM each in addition to 2.5U/100 μL of DNA polymerase (Hot Star Taq. Qiagen). Ten-fold diluted DNA template was added to the reaction mix at a volume of 1 μL per tube. The cycling program comprised an initial denaturation step of 95 °C for 15 min, followed by 30 cycles of denaturation at 94 °C lasting 60 s each. The annealing temperature was 50 °C for 60 s, followed by elongation at 72 °C for 70 s and primer extension lasted 5 min at 72 °C. The amplified PCR products were analyzed on 1%
agarose gel. The DNA products were compared to a standard of 1 kb PlusLadderTM (Invitrogen, Eggenstein, Germany) loaded at a volume of 5 μL per well. Electrophoresis was facilitated by an electric current of 100 V for 60 min. The DNA products were digitally photographed using a transilluminating camera, BioDocAnalyze (Biometra Goettingen, Germany). Amplified PCR products were cleaned using QIAquick kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The DNA was finally eluted in 30 μL buffer and quantified using fluorescent dye, PicoGreen (MoBiTec., Goettingen, Germany) at a wavelength of 485 nm emitted by fluorometer at 530 nm, Labsystems Fluoroskan II (GMI, Albertville, Minnesota, USA).
2.2.4 Single-strand conformation polymorphism (SSCP) technique
The genetic band profiles were analyzed through the deployment of polymerase chain reaction based single-strand conformation polymorphism (PCR-SSCP) technique. Separation of the single-stranded DNA fragments was achieved on non-denaturing polyacrylamide-gel.
2.2.4.1 Reagents and equipments used in SSCP technique
A 25 mL polyacrylamid-gel was prepared by mixing 7.813 mL of 2 x MDE solutions to 2.5 mL of 10x TBE and 14.69 mL of double distilled water in a 50 mL beaker. The mixture was filtered through a polyether sulfone-membrane of 0.45 μm pore sizes fitted to a syringe. The mixture was degassed under a vacuum chamber for 2 min after which 10 μL of TEMED (C6H16N2) and 25 μL of 40% APS were added under constant stirring. The notched glass and thermostatic plates were each lined with 1 mL bind-silane and repel silane, respectively, (Pharmacia Amersham Biotech, Freiburg, Germany). The two plates were clamped onto each other leaving 1 mL space between them into which the gel was casted and a 26 tooth-comb fixed to create wells for loading DNA after polymerization. The polymerized gel was built onto the macrophore and run for 17 h at 400 V, 20 mA, 10 W.
2.2.4.2 Generation of the single-stranded DNA
Single-stranded DNA was generated from purified amplified PCR products digested by lambda-exonuclease (Pharmacia Amersham Biotech, Freiburg, Germany). The reaction mix was incubated at 37 °C for 45 min once again purified using MiniElute kit (Pharmacia (Schweiger and Tebba, 1998; 2000).
of SSCP loading buffer was added to the purified single stranded DNA and denatured at 95 °C for 2 min then cooled for at least 3 min on ice. About 5 μL of denatured single strand DNA was loaded into each well and run under conditions described above.
2.2.4.3 SSCP gel silver staining and development
After running the gel for 17 h, the notched glass plate was unclamped, separated and placed into a basin filled with 500 mL of 10% acetic acid and swivelled at a speed of 20 rpm for 30 min during fixation. The plate was washed thrice in deionised water for 5 min each time after which the gel was stained for 30 min in silver nitrate solution (500 mL double distilled water, 0.5 g AgNO3 and 0.75 mL of 37% formaldehyde). The gel was developed in cold sodium carbonate deca-hydrate solution (600 mL double distilled water, 33.75 g sodium carbonate deca-hydrate, 0.6 mL of 0.2% sodium thiosulphate and 1.2 mL of 37% formaldehyde solution). Gel development was carefully performed and as soon as the bands became visible the reaction was stopped by removing the notched glass plate from developer solution and placing it once again into acetic acid. After 2 min, it was removed from acetic acid and placed in a water-filled basin for 1 h and finally air-dried under a fume-hood and later scanned. The scanned gel photos were then transformed using Corel Draw.
2.2.4.4 Band selection, excision and soaking
The developed bands were carefully identified and representatives chosen for cloning and sequencing. Band dominance (strongly staining), uniqueness (specific to treatments/zones) and commonness (uniformly found across treatments/zones) were the criteria used for their selection. The selected bands were excised and covered with a drop of PCR water to ease peeling off from the gel plate. Excised band slices were placed in PCR tubes then transferred into 50 μL crush and soak buffer comprising 0.5 M ammonium acetate (10 mM Mg2+-acetate, 1 mM EDTA [pH 8.0], and 0.1% sodium dodecyl sulphate). Band slices were soaked in buffer and incubated at 37 °C for 3 h while spinning at 550 Umin-1 on a thermomixer. About 40 μL of the solution was pipetted into a two-fold volume (80 μL) ethanol, precipitated at -20 °C over night and centrifuged at 130,000 x g for 25 min at 4 °C and the resulting supernatant was discarded whereas the pellet was dried at 37 °C for 15 min in a thermomixer before dissolving in previously incubated 12 μL of 10 mM Tris-EDTA buffer for 1 h. The extract was used to perform 50 μL PCR to amplify the DNA. The PCR
(Bäckman al., 2003).
product was checked on 1% agarose gel, purified by ‘Nucleospin Extract II’ (Macherey & Nagel, Dueren, Germany) following manufacturer’s protocol and ligated and transformed.
2.2.5 DNA cloning (ligation and transformation)
Gene inserts of reference clones were amplified with vector-specific primers. The amplicons were cloned into pGEM®-T-Easy vector (Promega, Mannheim, Germany) using TA cloning kit. Ligation was performed in 0.5 μL T 4 DNA-Ligase, 0.5 μL pGEM-T-Easy Vector and 2.5 μL buffer of 3.5 μL reaction mix per 1.5 μL DNA template. The mixture was hand shaken and incubated overnight at 4 °C. Two controls, positive (with insert) and negative control (distilled water) were included to help assess success or contamination of the ligated products. Transformation was achieved by incubating 2 μL of the ligated DNA and 36 μL of competent SM 109 cells for 20 min. Competent cells were heat-shocked for 45 s at 42 °C and immediately cooled for 2 min on ice. A ratio of 1:20 dilution of room temperature SOC medium was added then spinned at 600 rpm for 90 min while incubating at 37 °C. About 100 μL of the transformant was plated twice onto LB/ampicillin/X-Gal/IPTG/agar (LAXI) using sterile plating-rod on a clean bench and incubated at 37 °C overnight.
Nutrient agar was prepared from 2.5 g NaCl, 2.5 g yeast extract, 5 g trypton and 7.5 g agar and poured into Petri-dishes on a clean-bench. The plates were incubated overnight for 16-24 h at 37 °C. The resulting bacterial white cell colonies (with vector inserts) and blue cell colonies (without inserts) were assessed. Blue cell colonies were ignored during the selection of three separate white cell colonies for plating onto agar nutrient plates for further multiplication. A portion of the cell colonies on master plate were picked and lyzed in 50 μL buffer prepared from 0.05 M NaOH and 0.025% SDS. The cells were incubated for 15 min at 95 °C at a speed of 600 min-1on a thermomixer. 450 μL solution of sterile double distilled water was added, vortexed and centrifuged for 4 min at 10,000 rpm. The product was used for 25 μL PCR amplification. Upon amplification, 5 μL of amplified PCR product was checked on 0.8% agarose gel while the remaining was purified using Qiagen kit and eluted with 30 μL EB buffer. About 14 μL of the DNA was digested in 16 μL lamda-exonuclease at 37 °C for 45 min. The digested product was purified using Qiagen mini Elute kit and finally eluted with 10 μL EB buffer. About 9 μL bromophenol blue loading dye was added to the eluted DNA at a ratio of 1:1 (vol/vol). Clone and genomic DNA were concurrently run
on the same SSCP gel plates for easy comparison. Clone bands of similar migration positions to the excised genomic bands were chosen for sequencing.
2.2.6 DNA Sequencing
Selected clone bands were sequenced in MWG Licor sequencer (Biotech) using Epicentre-Kit.
2.2.6.1 Sequencing reagents and equipment
A 41 cm sequence gel was prepared by mixing 16.8 g urea, 4 mL of 10x TBE long run and 6 mL of 40% Rapid Gel then filled up with distilled water up to 40 mL mark, dissolved and filtered into a syringe fitted with 0.45 μm perforated membrane then degassed for 2 min. Upon degassing, 40 μL of TEMED and 72 μL APS were added while constantly stirring then casted into a sloppy screw-clamped gel plate. A fine toothed comb was fixed to create wells during the 2 h polymerization. The plate was fitted onto a sequencer and both upper and lower chambers were filled up with 1x TBE long run solution. The cloned DNA material was loaded and run for 17 h.
2.2.6.2 Sequencing procedure
Sequencing was performed in MWG Licor sequencer (Biotech) using Epicentre-Kit. Master mixes were prepared from 7.2 μL buffer, 4.8 μL distilled water, 1.0 μL polymerase and infrared labelled 1.0 μL forward/ reverse primers. Aliquots of 14 μL prepared in 0.5 mL tubes using Sequitherm ExcelTM II DNA sequencing kit-LC, Epicentre Biotechnologies. Each of the 14 μL master mixes and 3 μL DNA templates were spinned. Aliquots of 4 μL of forward /reverse master mixes were transferred into each of the four termination mixes then spinned shortly. 2 μL termination mixes of adenine, cytosine, guanine and th amine (A, C, G, T) were pipetted into 96 well-plate and shortly spinned. A drop of mineral oil (Sigma) was added to both reaction mixes and run for 90 min on M-13 Forward thermocycling programme. After amplification, 2 μL of stop-loading buffer was added, spined and denatured for 2 min at 95 °C and cooled for 3 min at 4 °C. 1 μL of the denatured product was loaded for sequencing and run for 17 h. The sequences were retrieved, processed and blasted in NCBI (Altschul et al., 1990). They were aligned in Mega-Program (Kumar et al., 2004) and imported into ARB-program for analysis and treeing (Ludwig et al., 2004).
2.2.7 Statistical Analyses
The SSCP gels were scanned and images adopted to analyze banding profiles in GelCompar® II programme package, version 4.5 of 2005 (Applied Maths BVBA, Belgium).
2.2.7.1 Digital image analysis
Each of the scanned SSCP gel images was transformed in Corel Draw. The banding lanes on each gel were automatically searched. Banding strips in each lane were evaluated on the basis of the corresponding densitometric tone curve values. Mobility of the band profiles were normalized with four external bacterial standards comprising Bacillus licheniformis, Rhizobium trifolii, Flavobacterium johnsoniae and Rhizobium radiobacte. They appeared in the same order in which they have been listed here above and were represented by I, II, III and IV on the marker lane upon migration on the gel. Band and reference positions were automatically aligned. In case the reference positions were not properly matched during the automatic alignment, internal standards were introduced to reorganize band positioning. The edited digital image was saved and desired gel strips were highlighted to calculate similarity matrices of the constructed dendrogram based on unweighted pair group method with arithmetic averages (UPGMA) and pearson correlation coefficients. Algorithms of the pearson’s correlation coefficients were utilized to compare nitrogen treatment effects of the corresponding banding profiles. Permutation test was performed to determine the differences among gel and treatment profiles whereas significant differences among band profiles were determined as described by Kropf et al. (2004) in SAS program.
2.2.7.2 Sequence analyses
Nucleotide sequences were determined by the Sanger method (Sanger et al., 1977). Clone sequences were processed as described by Ewing and Green, (1998) and Ewing et al. (1998). The sequences were compared with complete EMBL database nucleotide sequences by blasting as described by Altschul et al. (1990). Sequences with best BLAST scores were imported into ARB-program for treeing and analysis. Phylogenetic trees were constructed in ARB program for analysis (http:/www.arb-home.de) (Ludwig et al., 2004). Nearly full-lengths of sequences (>1000 bases) obtained were aligned using Mega-software then imported to ARB. Imported sequences were automatically aligned withexisting 16S rRNA
sequences in ARB alignment tool, ARB_EDIT, visually inspected and manually edited where necessary. Phylogenetic placement was achieved by comparing reference and obtained sequences in the domain bacteria. Ambiguous base positions were excluded during sequence similarity calculations. Overall phylogenetic analyses were determined by distance matrix, maximum parsimony and maximum likelihood methods as described by Ludwig and Schleifer (1999) and Ludwig et al. (1998). Statistical significance levels of interior nodes were determined by performing bootstrap analyses of neighbour joining method combined with Jukes-Cantor correction to infer distance matrix trees. Variability of individual alignment positions were determined in ARB_SAI tool based on remove or include criterion.
2.3 Evaluation of the biodiversity of ammonia oxidizing bacteria
2.3.1 Detection of ammonia oxidizing bacteria (AOB)
Soil samples for ammonia oxidizing bacteria (AOB) analysis were similar to those used to investigate bacterial community biodiversity and responses to CULTAN fertilization (section 2.2). Only vertical zones, 2a and 3 of the five nitrogen treatments were considered. No lateral zone was included. Treatment and sampling zone details as well as DNA extraction were as described in sections 2.2.1 and 2.2.2.
2.3.2 Selection and optimization of amoA primers
A review of published primers was made to select amoA primer pairs. Primer pair developed by Rotthauwe et al. (1997) was chosen. Forward primers targeting positions 332 to 349, (amoA-F; 5’–GGGGTTTCTACTGGTGGT- 3’) and reverse primer (amoA-R+Ph; 5’ –CCCCTCKGSAAAGCCTTCTTC- 3’) {K=G or T; S=G or C} targeted the stretch between 802 and 822 of the open reading frame of amoA gene sequence of Nitrosomonas europaea. The primers generated 491 bp length PCR products. Different concentrations were tested and 30 pmol μL-1 was adopted. Additional 1.2 μL of MgCl2 was applied to the reaction mix.
2.3.3 Selection of AOB for use as positive control
Amplification efficiency of the selected amoA primer set was tested on Nitrosospira multiformis, ATCC acquired from Jena University. The obtained amoA band size was 491 base pair length. N. multiformis clones were reamplified and checked on 1% agarose and yielded a similar size to the original plamid DNA. One of the five clones was adopted as a comparison check for amoA bands.
2.3.4 PCR amplification of AOB with amoA primers
Amplification of amoA genes were performed on primus thermocycler (MWG Biotech, Ebersberg, Germany) in 50 μL tube (Flat Cap Micro Tubes). The reaction mixture comprised 1× PCR buffer containing 1.5 mM MgCl2, to which 1.2 mM MgCl2was added, besides deoxynucleoside triphosphate (dNTPs) of 0.2 mM, forward and reverse primers of (0.3 μM each) as well as 2.5U/100 μL of DNA polymerase (Hot Star Taq, Qiagen). To this, 2 μL of 10-fold diluted template DNA was added in 50 μL reaction mix for amplification.
The cycling programme comprised an initial denaturation step of 95 °C for 15 min, followed by 36 cycles of denaturation at 94 °C lasting 90 s each. Primer annealing temperature was 60 °C for 90 s, followed by elongation at 72 °C for 90 s. The final extension step at 72 °C lasted 5 min. The PCR products were electrophorized and compared on 1% agarose to standards of 1 kb PlusLadderTM (Invitrogen, Eggenstein, Germany). The PCR products were amplified, purified and quantified as described in section 2.2.3 and digested to generate single-stranded DNA following the procedure outlined in section 2.2.4.2.
2.3.5 Development of amoA SSCP gels
Single-strand conformation polymorphism gels were developed from amoA genes based on the same procedure as described in section 2.2.4.
2.3.6 Band selection, ligation, transformation and sequencing
Both unique and common bands were selected, excised, ligated, transformed and sequenced as described in sections 2.2.5 to 2.2.6.
2.3.7 Statistical analysis
Statistical analyses of digital gel images were performed using Gelcompar software programme as described in section 2.2.6.1 and the generated similarity matrices were used for permutation test. Phylogenetic tree was constructed in ARB program using databank published amoA sequences.
2.4 Ammonia oxidizing bacteria population abundance in CULTAN-fertilized soils
2.4.1 Quantification of N. multiformis gene copies
Three separate PCR assays were performed to amplify N. multiformis amoA genes. The three PCR product replications were purified using QIAquick kit (Qiagen, Hilden, Germany) and eluted in 30 μL buffer. The products were pooled, mixed and quantified using fluorescent dye, PicoGreen (MoBiTec., Goettingen, Germany). Serial dilution ranges of 10 -2
to 10-10 were performed and measured by fluorometer, Labsystems Fluoroskan II (GMI, Albertville, Minnesota, USA) at 485 nm wavelength emitted at 530 nm. The measured DNA concentration was employed to calculate gene copy numbers. The amplification curve was normalized and threshold cycle (Ct) determined and used to calibrate gene copy numbers. Equation 1 outlines the calculation of standard gene copy numbers while equation 2 gives the DNA weight. When equation 2 is substituted into 1, equation 3 is formed. The new equation can be adopted to estimate standard gene copy numbers. Two constants were used, 6.023 x 1023was Avogadro’s constant whereas 6.6 x 1011 was the fragment base pair length of DNA strand.
Equation 1:
Standard copy numbers μL-1 = DNA concentration [ng . μL-1] DNA weight [ng . copy -1 ]
Equation 2:
DNA weight [ng . copy -1 ] = Fragment length [bp] x 6.6 x 1011 ng.[mol-1.bp]-1) 6.023 x 1023 (Copies. mol-1)
Equation 3:
Standard copy numbers μL-1 = DNA concentration [ng . μL-1] x 6.023 x 1023 (Copies. mol-1) Fragment length [bp] x 6.6 x 1011 ng.[mol-1.bp]-1)
2.4.2 Real-time PCR optimization
Real-time PCR assay was performed in 20 PL reaction mixture consisting of 1 ng template DNA, 10 PL of QauntiTech SYBR Green master mix (Qiagen) and 30 pmol PL-1 of
forward and reverse amoA primers. Non-coloured 50 PL flat-capped PCR micro tubes were used. The PCR cycling protocol adopted for amoA quantification was as follows: initial denaturation step at 95 °C for 15 min, followed by 50 cycles of denaturation at 94 °C lasting 60 s each. Primer annealing was at 60 °C for 60 s, elongation at 72 °C for 60 s and a final extension step at 72 °C for 5 min. Generated curves were analyzed to determine the threshold cycle for each assay.
2.4.3 Generation of amoA standard curves
Curves generated from selected ammonia oxidizing bacterial species, N. multiformis were used as standard AOB species to estimate the abundance of amoA genes in soil genomic DNA. The standard curves were developed through the performance of serial dilutions ranging between 10-3 and 10-7. The diluted positive control DNA ranged between 10-3 and 10-7. Along side the positive control were soil sample DNA template and non-template control (NTC). The amplified PCR products were electrophorisized and compared on 1% agarose to standards of 1 kb PlusLadder TM (Invitrogen, Eggenstein, Germany).
2.4.4 Threshold cycle determination
Threshold cycles of serially diluted N. multiformis were used to estimate amoA gene copy numbers of bacterial genomic DNA of the nitrogen treatments.
2.4.5 Melting point curves of amoA genes
The melting point (MP) of amoA genes was continuously recorded during the amplification. While some MP peaks of the soil extracted DNA corresponded to those of the standard AOB, a few did not. Given that melting point is a proof of purity, those with similar peaks indicated PCR product similarity, whereas dissimilar peaks suggested impurity or presence of a different product.
2.4.5 Statistical analysis
No statistical analysis was performed on the generated Real-time PCR data because
population estimates.
replicate test sample assays and non-template control (NTC) curves overlapped. Because of this limitation, no replicate quantification assays were factored to calculate ammonia oxidizing bacteria
2.5 Effect of temperature on CULTAN-fertilized barley
2.5.1 Experimental site and crop growth conditions
The experiment was performed in three separate growth chambers at Federal Agricultural Research Centre (FAL), Braunschweig, Germany. Spring barley, Hordeum vulgare L. cv. Maresi was used as a model cereal in the study. The seeds were sown singly within an inter- and intra- row spacing of 5 cm at a depth of 2.5 cm in 90 L soil filled container. The soil was a mixture of top-, sub- and sandy-soil proportions in the ratio of 1:1:1, respectively. It was free-draining sandy loam comprising 14.6% clay, 39.6% silt, 45.8% sand and 1.6% humus with a near neutral pH of 7.4. The total nitrogen and carbon were 0.03% and 0.43%, respectively. Growth chambers were cubes measuring 3 m (27 m3) fitted with 16 lamps on two horizontally adjustable metallic frames. Half of the lamps comprised sodium bulbs while the other eight were fluorescent potassium tubes. The two lamp sets produced 600 μmol m-2s-1 of photosynthetic active radiation (PAR) during the 14/10 h day/night photoperiod. Chamber relative humidities ranged between 55 – 70% and irrigation was regularly provided by hand held sprinkler. Five 1 cm diameter holes at the bottom of container drained excess water to guard against logging. The draining percolate was collected and reutilized in subsequent irrigation schedules.
2.5.2 Experimental design
Temperature was the major treatment whereas nitrogen was minor. Three temperature regimes LTR, MTR and HTR were established and within each growth temperature comprised three nitrogen treatments, ammonium, nitrate and non-fertilized control (Table 1). Each of the nitrogen treatments were replicated twice. The nitrogen treatments were arranged in a randomized complete block design (RCBD) within each temperature regime. The experiment was performed in two seasons under similar conditions, but chambers were swapped in each season to balance out their effects on crop growth.
2.5.3 Temperature and nitrogen treatments
Three days prior to sowing, all the growth chambers were acclimatized to the same growth conditions of 13/9 oC for d/n, respectively. This growth condition was maintained
